This invention relates to novel compounds that can complex with lanthanide cations processes for their preparation and the use of the resulting lanthanide chelates as biomolecular probes. In particular, this invention relates to complexing compounds which contain novel photosensitizers and can produce long-lived fluorescence for use in bioaffinity assays, especially those of HTRF (homogeneous time-resolved fluorescence).
With the growth of combinatorial chemistry and high-throughput screening, particularly within the pharmaceutical industry, the requirement for biological assays has dramatically increased. Traditional assay technologies, which are often based on radioisotope labels, are unable to achieve the desired throughput whilst simultaneously reducing assay volumes. As a result of the deficiencies inherent in traditional methodologies there has been a shift towards the use of new technologies based on fluorescence. Such techniques can have a number of advantages over radioactive assays, but ability to automate, ease of use, miniaturisability, and sensitivity are of particular importance. One of the primary technologies utilised is homogeneous time resolved fluorescence energy transfer (HTRF). This proximity based method requires the use of a fluorescent donor moiety covalently attached to the interacting molecules, either directly or via labelled antibodies or labelled streptavidin which, when in proximity with a second fluorescent or chromophoric label (the acceptor), leads to a modulation of the fluorescence properties of the donor. Such methods provide useful information about the structure, conformation, relative location and/or interactions of macromolecules. In particular. HTRF has widespread application in high throughput screening of molecular interactions and enzymes using proteins, ligands and substrates labelled with donors and acceptors.
Traditional fluorescent labels of organic dyes such as fluoresceins and rhodamines have long been employed as bioanalytical tools in immunoassays. Lanthanide chelates are more recently developed fluorescence agents and have been found to possess properties which make them very suited as potential labels in the bioassay field. Thus, the lanthanide chelates are capable of giving long-lived and longer wavelength fluorescent emissions upon excitation. Through time-delay measurements they have demonstrated clear advantages over conventional fluorescent labels in terms of experiencing less quenching and background interference while exhibiting increased detection sensitivity. In addition to these advantages, many lanthanide chelates have improved solubility properties and are able to efficiently transfer energy from their excited states to neighbouring acceptor molecules. As such they are ideal agents for HTRF use especially for developing high-throughput automated and miniaturized binding assays with the inclusion of immunoassays, DNA hybridization assays, receptor binding assays, enzyme assays. cell-based assays. immunocytochemical or immunohistochemical assays.
Lanthanide chelates typically comprise a chelating group which binds the lanthanide and an organic sensitiser group. The sensitiser group has the function of absorbing light and transferring energy to the lanthanide. It thereby overcomes the inherently low absorbance of the lanthanide ions. Such chelates have been extensively reviewed, for example in Li and Selvin (J. Am. Chem. Soc (1995) 117, 8132-8138). Lanthanide chelator groups comprising a plurality of polyaminocarboxylate groups are commonly used. European patent EP0203047B1 discloses fluorescent lanthanide chelates comprising xe2x80x9cTEKESxe2x80x9d (4-(4-isothio-cyanatophenylenthynyl-2,6-{N,N-bis(carboxymethyl)aminomethyl]-pyridine) type photosensitizers. Patent application WO 96/00901A1 discloses lanthanide chelates comprising the chelator group DTPA (diethylenetriaminepentacetic acid) covalently bonded to a coumarin or quinolone-like sensitisers. Heyduck and Heyduck (Anal. Biochem. (1997) 248, 216-227) describe compounds of similar structure to those of WO 96/00901 but differ in that they possess a thiol-reactive pyridyl disulphide moiety which allows covalent attachment to macromolecules.
It is widely recognised that the role of the sensitiser group is of fundamental importance in that they impart to the chelates different physicochemical properties pertaining to excitation wavelength, lifetime, quantum yield, quenching effect, complex stability, photostability, solubility, charge, nonspecific protein interaction, biocoupling efficiency and ease of preparation. It is advantageous to have a diversity of novel fluorescent probes to use and develop HTRF assays. There is consequently a need for more and better ways of fluorescently labelling assay components.
The present invention therefore provides, in a first instance, a lanthanide chelate comprising one or more sensitiser group(s) covalently attached to a lanthanide chelating group which is characterised in that the sensitiser group is a group of formula (I) 
where X is a group that couples the said sensitiser group to the said chelating group.
Suitably X is any group that is capable of covalently linking the sensitiser group with the chelator group and, at the same time, does not affect the ability of the chelating group to bind the lanthanide cation. Preferably X is a group xe2x80x94NH(CH2)pNHxe2x80x94 in which p is 2, 3 or 4 and which forms an amide bond with the chelating group. Most preferably X is a group xe2x80x94NH(CH2)2NHxe2x80x94.
Preferably the lanthanide chelate contains 1 or 2 sensitiser group(s) of formula (I).
Where used herein the term [lanthanide] chelating group is used to describe a group that is capable of forming a high affinity complex with lanthanide cations such as Tb3+, Eu3+, Sm3+, Dy3+. Any fluorescent lanthanide metal can be used in the chelates of this invention but it is expected that chelates containing europium or terbium will possess the best fluorescent properties. Most preferably the lanthanide metal is europium.
Suitable examples of chelating groups include those described in WO 96/00901. Preferably the chelating group will be either DTPA (diethylenetriaminepentacetic acid) or TTHA (triethylenetetraaminehexacetic acid), that is to say compounds of the formula (II) 
where n=1 (DTPA) or n=2 (TTHA). Both DTPA and TTHA are well known in the art and are available from commercial suppliers. Alternatively the chelating group is a compound of formula (III). 
A compound of formula (III) may be prepared by reaction of the corresponding N,N-xcex1-bis(carboxymethyl)-L-lysine with isophthalic acid activated by Oxe2x80x94(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate.
Typically a compound of formula (IV) 
in which L is a group xe2x80x94NH(CH2)pNH2 where p is 2, 3 or 4 would be used in the preparation of a lanthanide chelate of formula (I). A compound of formula (IV) can be prepared from Cinoxacin (commercial product supplied by Sigma) by forming its acid chloride derivative and then reacting with an appropriate alkylene diamine reagent. A compound of formula (IV) in which p is 3 or 4 is believed to be novel.
Compounds of structure (I) have desirable spectral properties in solution, but to use them in a biological assay it is necessary to attach them to the molecules such as proteins, nucleic acids, lipids, carbohydrates or peptides. Reagents containing reactive groups suitable for derivatising macromolecules will also form part of the invention. The invention therefore provides, in a further aspect, for a lanthanide chelate of structure (I) further comprising a linker group wherein the linker group is either a group of formula (V) 
in which Y is CH2, CH2CH2 or xe2x80x94CH2CH(COOH)xe2x80x94 and R2 is a reactive group which is suitable for derivatising macromolecules; or the linker group is a group of formula (VI)
R2xe2x80x94(CH2)nxe2x80x94Zxe2x80x94NHxe2x80x94xe2x80x83xe2x80x83(VI)
in which n is 1 to 5, Z is a bond or a group xe2x80x94CH2CH(COOH)xe2x80x94 and R2 is as defined for formula (V).
For groups of formula (V) and (VI) the point of attachment to the chelating group is via the amine functionality, thus forming an amide bond. Compounds of structure (V) and (VI) can be prepared from compounds of structure (I) by reaction of the mixed anhydride of (I) with the appropriate amine.
It will be appreciated by those skilled in the art that lanthanide chelates comprising linker groups of formula (V) or (VI) can, rather than labelling the target macromolecule directly, be alternatively used to label streptavidin or an antibody, which in turn binds to the target macromolecule. In such circumstances the group R2 contains an epitope for an antibody or a ligand for other proteins to be used for indirect bioconjugation.
Typically the group R2 is an amine reactive group, a thiol reactive group or a photoactivatable reactive group. Suitable examples of amine reactive group are those that can covalently couple with an amine functionality on a macromolecule and includes groups such as isothiocyanate (NCS) and the chlorotriazine of structure (VII); 
Suitable examples of thiol reactive group are those that can covalently couple with a thiol functionality on a macromolecule and includes groups such as iodoacetamide (xe2x80x94NHCOCH2I) and the maleimide of structure (VIII) 
Suitable examples of a photoactivitable reactive groups include the azide of structure (IX) or of structure (X). 
Conjugation of such chelates onto biomolecules are typically performed by incubating the reactive chelate, either in the absence or presence of the lanthanide ion, with the target molecule of interest, typically a biomolecule under conditions where the reactive groups of the target molecule are derivatised with the chelate. The reactions are terminated by quenching with an excess of a reagent with the same reactive functionality as the target molecule. If not already present in the chelate, excess lanthanide is added to the quenched reaction mixture. The purified target-chelate-lanthanide ion complex is typically obtained by preparative gel filtration, ion exchange, reverse phase or other chromatographic procedures to remove the excess chelate and, where appropriate, the lanthanide ion from the labelled target molecule.
HTRF assays are typically performed whereby the enzyme reaction or molecular interaction of interest is configured to ensure that the specific activity under investigation leads, either directly or indirectly, to a change in the mean distance between the lanthanide-chelate-target molecule and another molecule which, either itself or via conjugation to another entity (the acceptor), results in a modulation of the optical properties of the lanthanide ion or of the acceptor.
For example, target molecules are labelled directly with lanthanide chelates onto amine (Lys, N-termini), thiol (Cys), His, or Tyr residues in proteins or alternatively indirectly through antibodies, protein A or G, or streptavidin which are themselves labelled with lanthanide chelates. Acceptor molecules, such as reactive blue 4 or phycobiloproteins such as allophycocyanin, can be conjugated either directly onto the co-target of interest or indirectly via standard heterobifunctional cross linking chemistry onto, for example, streptavidin, antibodies or protein A/G.
The extent of energy transfer between the donor and acceptor can be determined either by monitoring changes in the radiative lifetime of the donor and/or acceptor e.g. using a time resolved fluorescence instrument or by measuring the time gated change in total sample intensity e.g. using a time gated fluorescence mictotitre plate reader.
The following Descriptions and Examples serve to illustrate the invention